Polyketides are a diverse class of compounds that are the source of many biologically active molecules such as tetracycline, erythromycin, epothilone, narbomycin, picromycin, rapamycin, spinocyn, and tylosin. Other important examples include naturally occurring immunosuppressants FK-506 (also known as tacrolimus) and FK-520 (also known as ascomycin). 
Differing by a single substituent (R1) at C-21, FK-506 has an allyl group whereas FK-520 has an ethyl group at this position. Of the two, FK-506 has been particularly well studied and is used currently as an immunosuppressive drug.
FK-506 and FK-520 exert their biologic effects through the initial formation of an intermediate complex with proteins known as FKBPs (FK-506 binding proteins) such as FKBP-12 and FKBP-52. These proteins are a class of cytosolic proteins that form complexes with molecules such as FK-506, FK-520, and rapamycin that in turn serve as ligands for other cellular targets involved in signal transduction. Binding of FK-506, FK-520, and rapamycin to FKBP occurs through the structurally similar segments of the polyketide molecules, known as the xe2x80x9cFKBP-binding domainxe2x80x9d (as generally but not precisely indicated by the stippled regions in the structures below). 
The FK-506-FKBP complex then binds calcineurin, while the rapamycin-FKBP) complex binds to a protein known as RAFT-1. Binding of the FKBP-polyketide complex to these second proteins occurs through the dissimilar regions of the drugs known as the xe2x80x9ceffectorxe2x80x9d domains.
The three component FKBP-polyketide-effector complex is required for signal transduction and subsequent immunosuppressive activity of FK-506, FK-520, and rapamycin. Modifications in the effector domains of FK-506, FK-520, and rapamycin that destroy binding to the effector proteins (calcineurin or RAFT) but leave FKBP binding unaffected lead to loss of immunosuppressive activity. Further, such analogs antagonize the immunosuppressive effects of the parent polyketides, because they compete for FKBP. Such non-immunosuppressive analogs also show reduced toxicity (see Dumont et al., 1992, Journal of Experimental Medicine 176, 751-760), indicating that much of the toxicity of these drugs is through a mechanism not mediated by FKBP binding.
In addition to immunosuppressive activity, FK-520, FK-506, and rapamycin have neurotrophic activity. In the central nervous system and in peripheral nerves, the corresponding target proteins are referred to as neuroproteins. The neuro-FKBP is markedly enriched in the central nervous system and in peripheral nerves. Molecules that bind to the neuro-FKBP, such as FK-506 and FK-520, have the remarkable effect of stimulating nerve growth. In vitro, they act as neurotrophins. More particularly, they promote neurite outgrowth in NGF-treated PC12 cells and in sensory neuronal cultures, and they promote regrowth of damaged facial and sciatic nerves, and repair lesioned serotonin and dopamine neurons in the brain in intact animals. See Gold et al., June 1999, J. Pharm. Exp. Ther. 289(3): 1202-1210; Lyons et al., 1994, Proc. National Academy of Science 91: 3191-3195; Gold et al., 1995, Journal of Neuroscience 15: 7509-7516; Steiner et al., 1997, Proc. National Academy of Science 94: 2019-2024; and U.S. Pat. Nos. 5,968,921 and 6,210,974. Further, the restored central and peripheral neurons appear to be functional.
Compared to protein neurotrophic molecules (e.g., BNDF, NGF, etc.), the small-molecule neurotrophins such as FK-506, FK-520, and rapamycin have different, and often advantageous properties. First, whereas protein neurotrophins are difficult to deliver to their intended site of action and may require intra-cranial injection, the small-molecule neurotrophins display excellent bioavailability; they are active when administered subcutaneously and orally. Second, whereas protein neurotrophins show quite specific effects, the small-molecule neurotrophins show rather broad effects. Finally, whereas protein neurotrophins often show effects on normal sensory nerves, the small-molecule neurotrophins do not induce aberrant sprouting of normal neurconal processes and seem to affect damaged nerves specifically. Neuro-FKBP ligands have therapeutic utility in a variety of disorders involving nerve degeneration (e.g. multiple sclerosis, Parkinson""s disease, Alzheimer""s disease, stroke, traumatic spinal cord and brain injury, peripheral neuropathies).
The metabolism and pharmacokinetics of FK-506 have been extensively studied, and FK-520 is believed to be similar in these respects. Absorption of FK-506 is rapid, variable, and incomplete from the gastrointestinal tract (Harrison""s Principles of Internal Medicine, 14th edition, 1998, McGraw Hill, 14, 20, 21, 64-67). The mean bioavailability of the oral dosage form is 27% (range 5 to 65%). The volume of distribution (VolD) based on plasma is 5 to 65 L per kg of body weight (L/kg), and is much higher than the VolD based on whole blood concentrations, the difference reflecting the binding of FK-506 to red blood cells. Whole blood concentrations may be 12 to 67 times the plasma concentrations. Protein binding is high (75 to 99%), primarily to albumin and alpha1-acid glycoprotein. The half-life for distribution is 0.9 hour; elimination is biphasic and variable: terminal-11.3 hours (range, 3.5 to 40.5 hours). The time to peak concentration is 0.5 to 4 hours after oral administration.
FK-506 is metabolized primarily by cytochrome P450 3A enzymes in the liver and small intestine. The drug is extensively metabolized with less than 1% excreted unchanged in urine. Because hepatic-dysfunction decreases clearance of FK-506, doses have to be reduced substantially in primary graft non-function, especially in children. In addition, the bioactivity of FK-506 is affected by drugs that modulate the activity of P450 3A enzymes. Drugs that induce the cytochrome P450 3A enzymes reduce FK-506 levels, while drugs that inhibit these P450s increase FK-506 levels. For example, FK-506 bioavailability doubles with co-administration of ketoconazole, a drug that inhibits P450 3A. See, Vincent et al., 1992, Arch. Biochem. Biophys. 294: 454-460; Iwasaki et al., 1993, Drug Metabolism and Disposition 21:971-977; Shiraga et al., 1994, Biochem. Pharmacol. 47: 727-735; and Iwasaki et al., 1995, Drug Metabolism and Disposition 23: 28-34.
FIG. 1 shows the eight isolated metabolic products formed from incubation of FK-506 with liver microscomes. As can be seen, four metabolites of FK-506 involve demethylation of the methoxy groups on carbons 13, 15, and 31, and hydroxylation of carbon 12. The 13-demethylated (hydroxy) compounds undergo cyclizations of the 13-hydroxy at carbon 10 to give M-I, M-VI and M-VII, and the 12-hydroxy metabolite at carbon 10 to give M-I. Another four metabolites formed by oxidation of the four metabolites mentioned above were isolated by liver microsomes from dexamethasone treated rats. Three of these are metabolites doubly demethylated at the methoxy groups on carbons 15 and 31 (M-V), 13 and 31 (M-VI), and 13 and 15 (M-VII). The fourth, M-VIII, was the metabolite produced after demethylation of the carbon 31-methoxy group, followed by formation of a fused ring system by further oxidation. Among the eight metabolites, M-II has immunosuppressive activity comparable to that of FK-506, whereas the other metabolites exhibit weak or negligible activities. Importantly, the major metabolite of human, dog, and rat liver microsomes (representing approximately about 90% of the metabolic products after a 10 minute incubation) is the 13-demethylated and cyclized FK-506 (M-I).
A disadvantage of using FK-506 and FK-520 as drugs is dosing unpredictability. Due to the significant variability in metabolism among patients, an appropriate dosing regimen is difficult to ascertain for an individual patient. Another disadvantage of FK-506 and FK-520 is their dual pharmacological effects as immunosuppressants and as neurotrophic agents. In general, compounds having a single specificity are desired. For example, a FK-506 like compound having only neurotrophic activity without immunosuppressive activity or vice versa may be used to treat the intended symptom without the side effects of the other bioactivity.
As a result, derivatives that improve upon the properties of FK-506 and FK-520 are needed and desired. However, because FK-506 and FK-520 are complex structures that are generally not amenable to either de novo chemical synthesis or facile derivation, this need remains unfulfilled.
The present invention relates to novel polyketides, host cells that produce the novel compounds, and methods fort their use. The compounds of the present invention are cyclic polyketides (also referred to as a xe2x80x9cmacrolidesxe2x80x9d or xe2x80x9cmacrolactonesxe2x80x9d) that include 
as part of their structure and bind to a FK binding protein wherein R4 and R5 are each selected from the group consisting of hydrogen, methyl, ethyl, and methoxy, provided that at least one of R4 and R5 is hydrogen, methyl, or ethyl. As will be explained in greater detail below, the compounds of the present invention have properties such as favorable P450 enzyme activity profiles that are desirable for use of these compounds as drugs.